p19 arf  (Novus Biologicals)

Journal: bioRxiv

Article Title: Multi-stromal organoid co-cultures model pancreatic cancer and pancreatitis epithelial cell-fibroblast heterogeneity

doi: 10.64898/2025.12.05.692494

Figure Lengend Snippet: (A) Schematic of flow sorting strategy of organoid and pancreatic stellate cell (PSC) monocultures (monocx) and P or PDAC organoid/PSC co-cultures (cocx) in reduced media (i.e. 5% FBS/DMEM) prior to RNA-seq. (B) Representative images of H&E, podoplanin (PDPN) and CK19 stains of P or PDAC organoid/PSC cocx after 7 days in culture. Scale bars = 100 μm. (C) Fold change in numbers of PSCs (PDPN + EpCAM - ) flow-sorted from monocultures (monocx) or from P or PDAC cocx after 3.5 days in culture relative to numbers of PSCs plated at day 0. Results show mean ± SEM. ** P adj < 0.01; *** P adj < 0.001, Kruskal-Wallis test. (D) Fibroblast (PDPN + EpCAM - )/epithelial cell (EpCAM + PDPN - ) ratio as assessed by flow cytometry of P or PDAC organoid/PSC cocx after 3.5 days in culture. Results show mean ± SEM. * P < 0.05, Mann-Whitney test. (E) Significantly upregulated (i.e., normalised enrichment score, NES > 1.50 and FDR < 0.25) and downregulated (i.e., NES < -1.50 and FDR < 0.25, apart for TGF-β-signaling KEGG with NES = -1.496) pathways identified by GSEA of PDAC organoids in monocx or cocx compared to P organoids in monoculture or co-culture, respectively, as assessed by RNA-seq. (F) Venn diagrams of curated protein-coding genes significantly ( P adj < 0.05) upregulated in P organoids in PSC co-culture compared to PDAC organoids in PSC co-culture (hereon, ‘P organoid markers from PSC co-cultures) and murine pancreatitis Ductal 1 or Ductal 2 cells compared to PDAC malignant cells in vivo (hereon, ‘pancreatitis Ductal 1 markers’ and ‘pancreatitis Ductal 2 markers’, respectively), as assessed by RNA-seq and snRNA-seq, respectively. Significance of the overlap between datasets was defined by hypergeometric test (with denominator n=18,000). Selected genes common to two or more datasets are indicated. (G) Heatmap of scaled expression of selected genes differentially upregulated in murine pancreatitis epithelial clusters. Data are scaled such that the cluster with the lowest average expression = 0 and the highest = 1 for each gene. (H) Western blot analysis of SOX9 in P and PDAC organoids cultured in reduced media for 3 days. ACTIN, loading control. (I) Venn diagrams of curated protein-coding genes significantly ( P adj < 0.05) upregulated in murine PDAC malignant cells compared to pancreatitis ductal cells in vivo (hereon, ‘PDAC malignant markers’) and murine PDAC organoids in PSC co-culture compared murine P organoids in PSC co-culture (hereon, ‘PDAC organoid markers from PSC co-cultures’), as assessed by snRNA-seq and RNA-seq, respectively. Significance of the overlap between datasets was defined by hypergeometric test (with denominator n=18,000). Selected genes common to two or three datasets are indicated. (J) Heatmap of scaled expression of genes differentially upregulated in murine PDAC epithelial clusters. Data are scaled such that the cluster with the lowest average expression = 0 and the highest = 1 for each gene. (K) RNA-seq expression of Cdkn2a-201 (encoding p16) and Cdkn2a-202 (encoding p19) in P or PDAC monocx and cocx with PSCs. Results show mean ± SEM. * P adj < 0.05; ** P adj < 0.01; *** P adj < 0.001, Kruskal-Wallis test. (L) Western blot analysis of p19 and p16 in P and PDAC organoids cultured in reduced media for 3 days. ACTIN, loading control. (M) RNA-seq expression of Tgfbr1 and Sparc in P or PDAC organoid monocx and P or PDAC cocx with PSCs. Results show mean ± SEM. * P adj < 0.05; ** P adj < 0.01; *** P adj < 0.001, Kruskal-Wallis test. (N) RNA-seq expression of Top2a and Dusp6 in P or PDAC monocx and cocx with PSCs. Results show mean ± SEM. ** P adj < 0.01; *** P adj < 0.001, Kruskal-Wallis test.

Article Snippet: Primary antibodies used were ACTIN (8456; Cell Signaling Technology; RRID:AB_10998774), p-STAT3 (9145; Cell Signaling Technology; RRID:AB_2491009), STAT3 (9139; Cell Signaling Technology; RRID:AB_331757), p16 (211542; Abcam; RRID:AB_2891084) and p19 (77184; Cell Signaling Technology), and SOX9 (AB5535; Merck; RRID: AB_2239761).

Techniques: RNA Sequencing, Flow Cytometry, MANN-WHITNEY, Co-Culture Assay, In Vivo, Expressing, Western Blot, Cell Culture, Control

Journal: bioRxiv

Article Title: Multi-stromal organoid co-cultures model pancreatic cancer and pancreatitis epithelial cell-fibroblast heterogeneity

doi: 10.64898/2025.12.05.692494

Figure Lengend Snippet: (A) Representative images of PDGFRα stains of P and PDAC organoid/pancreatic stellate cell (PSC) co-cultures after 7 days in culture. Scale bars = 100 μm. (B) Bright field images of P and PDAC organoid/PSC co-cultures after 7 days in culture. Scale bars = 100 μm. (C) Fibroblast (PDGFRα + )/epithelial cell (CK19 + ) ratio as assessed by immunohistochemical analysis of pancreatitis or PDAC murine tissues (from ). Results show mean ± SEM. *** P < 0.001, Mann-Whitney test. (D) Ratio of fibroblast to epithelial cell numbers per sample as determined by snRNA-seq analysis of pancreatitis and PDAC murine tissues (from ). Results show mean ± SEM. ** P < 0.01, Mann-Whitney test. (E) Summary table of the conditions analysed by RNA-seq in monoculture (i.e. culture of a single cell type) or co-culture (i.e. culture of two or more cell types). (F) PCA of P organoids in monocultures (i.e., P monocx, n=5 biological replicates, each with 2 replicates of different passages) or in co-culture with PSCs (i.e., P cocx, n=10 biological replicates), PDAC organoids in monocultures (i.e., PDAC monocx, n=4 biological replicates, of which three samples with 2 replicates of different passages) or in co-culture with PSCs (i.e., PDAC cocx, n=7 biological replicates), and PSC monocultures (i.e., PSC monocx, n=2 biological replicates, each with 2 replicates of different passages) and in co-culture with PDAC organoids (i.e., PDAC PSCs, n=7 biological replicates) or P organoids (i.e., P PSCs, n=10 biological replicates), as assessed by RNA-seq. (G) Significantly upregulated and downregulated pathways identified by GSEA of P or PDAC organoids in co-culture compared to their respective monocultures, as assessed by RNA-seq. (H) Heatmap showing GSVA enrichment scores (averaged per group) of selected pathways in P and PDAC organoid monocultures and co-cultures, as assessed by RNA-seq. Pathways displayed correspond to pathways significantly altered in GSEA ( and/or S4G). (I) GSEA of the murine pancreatitis Ductal 1 (top) and Ductal 2 (bottom) signatures in P organoids in co-culture with PSCs compared to P organoids in monoculture. The pancreatitis Ductal 1 signature was significantly enriched in P organoids in co-culture. The pancreatitis Ductal 2 signature was not significantly different between conditions. (J) Venn diagrams of differentially expressed curated protein-coding ortholog genes significantly ( P adj < 0.05) upregulated in P organoids in PSC co-cultures compared to PDAC organoids in PSC co-cultures (hereon, ‘P organoid orthologs from PSC co-cultures’), as assessed by RNA-seq, murine pancreatitis ductal orthologs, and human pancreatitis ductal orthologs. Selected genes common to all or two datasets are indicated. Significance of the overlap between P organoid orthologs from PSC co-cultures and human pancreatitis ductal orthologs was defined by hypergeometric test (with denominator = 16,076 orthologs). (K) Venn diagrams of differentially expressed curated protein-coding ortholog genes significantly ( P adj < 0.05) upregulated PDAC organoids in PSC co-cultures compared to P organoids in PSC co-cultures (hereon, ‘PDAC organoid orthologs from PSC co-cultures’), as assessed by RNA-seq, murine PDAC malignant orthologs, and human PDAC malignant orthologs. Selected genes common to all or two datasets are indicated. Significance of the overlap between PDAC organoid orthologs from PSC co-cultures and human PDAC malignant orthologs was defined by hypergeometric test (with denominator = 16,076 orthologs). (L) Western blot of p19 in N and PDAC organoids, as well as PDAC organoids that have not undergone loss of heterozygosity of the wild-type Trp53 allele (KPC organoids) and pre-cancerous pancreatic intraepithelial neoplasia organoids from KC mice (KC organoids) cultured in complete media for 3 days. ACTIN, loading control. (M) Venn diagrams of curated protein-coding significantly ( P adj < 0.05) upregulated genes in P organoids in co-culture with PSCs compared to PDAC organoids in co-culture with PSCs (hereon, ‘P organoid markers from PSC co-cultures’) and curated protein-coding significantly upregulated genes in P organoids in co-culture with PSCs compared to P organoids in monoculture. (N) Venn diagrams of curated protein-coding significantly ( P adj < 0.05) upregulated genes in PDAC organoids in co-culture with PSCs compared to P organoids in co-culture with PSCs (hereon, ‘PDAC organoid markers from PSC co-cultures’) and curated protein-coding significantly upregulated genes in PDAC organoids in co-culture with PSCs compared to PDAC organoids in monoculture.

Article Snippet: Primary antibodies used were ACTIN (8456; Cell Signaling Technology; RRID:AB_10998774), p-STAT3 (9145; Cell Signaling Technology; RRID:AB_2491009), STAT3 (9139; Cell Signaling Technology; RRID:AB_331757), p16 (211542; Abcam; RRID:AB_2891084) and p19 (77184; Cell Signaling Technology), and SOX9 (AB5535; Merck; RRID: AB_2239761).

Techniques: Immunohistochemical staining, MANN-WHITNEY, RNA Sequencing, Co-Culture Assay, Western Blot, Cell Culture, Control

Journal: Cancers

Article Title: Derivation of Genetically Defined Murine Hepatoblastoma Cell Lines with Angiogenic Potential

doi: 10.3390/cancers17183002

Figure Lengend Snippet: Properties of BN and YN HBs and their progeny immortalized cell lines. ( A ) Gross appearance of typical tumors generated by the indicated combinations of oncogenic drivers. Images of previously generated BY and BYN tumors are included for comparison. To allow for the establishment of immortalized BN and YN cell lines, all tumors were generated with the inclusion of 2 Crispr/Cas9 vectors encoding 4 different gRNAs directed against exon 2 of the Cdkn2a locus . ( B ) H&E-stained sections of tumors from ( A ). Note the more prominent blood vessels in BN tumors (black arrow) and the previously reported fluid-filled cysts adjacent to regions of necrosis in BYN tumors (black and green arrows, respectively) [ , ]. Scale bars = 500 μm. ( C ) H&E-stained cells from the indicated immortalized cell lines propagated on coverslips in vitro. BY1 and BYN2 cell lines were derived and characterized previously and are included here for comparative purposes . Scale bars = 50 μm. ( D ) Growth curves of the indicated cell lines and their suppression via the enforced expression of WT p16 INK4A and p19 ARF . The indicated cell lines were transfected with a control pSBbi-RP SB vector or with vectors encoding WT p16 INK4A or p19 ARF . Two days later, the cells were seeded into 12-well plates and maintained in 2 μg/mL of puromycin while monitoring dTomato expression. Subsequent growth was monitored using an Incucyte S3 Live-Cell imaging and Analysis System. Each point represents the mean of 4 replicas +/− 1 S.E. Note that BN2 cells were resistant to transfection on multiple occasions.

Article Snippet: Antibodies used included those directed against p16 INK4A (# Ab211542 , 1:2000, Abcam, Cambridge, UK), p19 ARF (#NB200-174, 1:1000, Novus Biologicals, Centennial, CO), GAPDH (#G8795, 1:10,000, Sigma-Aldrich, Inc. St Louis, MO, USA), YAP (#4912, 1:1000, Cell Signaling Technologies [CST], Inc., Danvers, MA, USA).

Techniques: Generated, Comparison, CRISPR, Staining, In Vitro, Derivative Assay, Expressing, Transfection, Control, Plasmid Preparation, Live Cell Imaging

Journal: Cancers

Article Title: Derivation of Genetically Defined Murine Hepatoblastoma Cell Lines with Angiogenic Potential

doi: 10.3390/cancers17183002

Figure Lengend Snippet: YN and BN cell lines express small, unique subsets of p16 INK4A and p19 ARF mutants. ( A ) Depictions of the proteins encoded by the 5 most abundant Cdkn2a mutations identified in each of the indicated cell lines. The frequencies with which they were detected based on deep sequencing of Cdkn2a exon 2 PCR products obtained from cell lines are indicated to the right of each cartoon. Asterisks indicate the mutants that were deemed the most likely to be expressed as proteins, based upon in silico translation of the open reading frames and the sizes of the actual proteins observed by immunoblotting (panel B ). contains a more comprehensive list of the mutations identified and their abundance. *: Proteins predicted to be expressed. ( B ) Immunoblots of mutant p16 INK4A and p19 ARF proteins expressed by the indicated cell lines. Included as controls were a sample of normal liver (L) and a primary BY HB (T) with intact Cdkn2a loci. The latter expressed WT p16 INK4A and p19 ARF as previously described . Expression of each protein relative to that of GAPDH, as determined by densitometric scanning, is shown beneath the blot. ( C ) BY-derived mutant and/or fusion p16 INK4A and p19 ARF proteins were used for expression in BN and YN cell lines. All encoded proteins were V5 epitope-tagged to allow expression levels to be directly compared. See ref. for previous characterization. ( D ) Approach to evaluating the growth suppressive effects of the mutants depicted in C on BN1 and YN2 cells. ( E ) Differential selection of p16 INK4A /p19 ARF mutants. The indicated cell lines were individually co-transfected with pSBbi SB vectors encoding the mutant proteins depicted in panel ( C ) plus an equal amount of the empty pSBbi vector. Two days later, half the cells were used to assess the transient expression of each protein as described in ( D ). The remaining cells were selected in puromycin for 14 and 21 days and assessed for the expression of their respective protein at these times using anti-V5 or anti-p16 INK4A antibodies. Expression of each protein relative to that of GAPDH, as determined by densitometric scanning, is shown to the right of the blot. ( F ) Selective retention of the non-resolvable mutants shown in ( E ). BN1 cells were separately co-transfected with the empty pSBbi vector alone plus one encoding each of the four indicated mutants. Puromycin-resistant clones were then selected and expanded for 3 weeks as in ( E ), followed by immunoblotting to detect each of the V5-tagged mutants. Expression of each protein relative to that of GAPDH, as determined by densitometric scanning, is shown beneath the blot. ( G ). The indicated vectors were transfected into YN2 or BN1 cells, which were then seeded into 12-well plates 2 days later in the presence of puromycin and enumerated over the course of the next 10 days. Each point represents the mean of 4 replicas +/− 1 S.E. Original Western Blot images in .

Article Snippet: Antibodies used included those directed against p16 INK4A (# Ab211542 , 1:2000, Abcam, Cambridge, UK), p19 ARF (#NB200-174, 1:1000, Novus Biologicals, Centennial, CO), GAPDH (#G8795, 1:10,000, Sigma-Aldrich, Inc. St Louis, MO, USA), YAP (#4912, 1:1000, Cell Signaling Technologies [CST], Inc., Danvers, MA, USA).

Techniques: Sequencing, In Silico, Western Blot, Mutagenesis, Expressing, Derivative Assay, Selection, Transfection, Plasmid Preparation, Clone Assay

Journal: Life

Article Title: Trophoblast Extracellular Vesicles as Modulators of Keratinocyte Stress Response and Senescence

doi: 10.3390/life15060918

Figure Lengend Snippet: Pretreatment of keratinocytes with trophoblast-derived extracellular vesicles (TEVs) prevents upregulation of senescence markers and cell cycle arrest. Bar graphs indications: ( A ) Expression of cyclin-dependent kinase inhibitors p19 and p21 in keratinocytes, as determined by cELISA. Cumulative data from two experiments (3 replicates) are shown as mean fold change relative to the unexposed cells (CTRL) + SD. ( B ) Representative overlaid flow cytometry histograms indicate the frequency of cells in G0/G1, S, G2/M phases of the cell cycle. Data (mean + SD) are representative from one of two experiments (4 replicates) with consistent results. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Briefly, after fixing the cells with ice-cold acetone/methanol (1:1) for 5 min and blocking with 1% bovine serum albumin (BSA) in PBS for 30 min, they were incubated with rabbit polyclonal anti-p19 ARF (hereafter: p19), anti-p21 CIP1/WAF1 (hereafter: p21), anti-hypoxia-inducible factor (HIF)-1α (all Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-integrin-β1(Merck KGaA, Darmstadt, Germany), and anti-Ki-67 (Sigma Aldrich, St. Louis, MO, USA) antibodies, diluted in 1% BSA/PBS at 4 °C overnight in a humidified chamber.

Techniques: Derivative Assay, Expressing, Flow Cytometry

Journal: JCI Insight

Article Title: BPDCN MYB fusions regulate cell cycle genes, impair differentiation, and induce myeloid–dendritic cell leukemia

doi: 10.1172/jci.insight.183889

Figure Lengend Snippet: ( A ) V5 and IgG CUT&RUN tracks at the Il3ra , Bcl2 , and Myc loci in Cdkn2a -KO MYB::PLEKHO1 leukemia cells. ( B ) TPM of Il3ra (CD123), Bcl2 , Myc , Ncam1 (CD56), Nrp1 (BDCA4), and Irf4 in Hoxb8-FL cell–derived pDCs ( n = 8) and Hoxb8-FL cell–derived leukemias of the indicated genotypes ( n = 4 per genotype). ( C ) Growth curves of Hoxb8-FL.MS5 cells transduced with V5-MYB constructs of the indicated genotypes. ( D ) Western blots showing p16INK4A and p19ARF expression in Hoxb8-FL.MS5 cells of the indicated genotypes. ( E ) Cell cycle analysis of Hoxb8-FL.MS5 cells. (Left) Representative flow plots of the indicated genotypes. (Right) Quantification of cells in each cell cycle phase as a proportion of total cells ( n = 4 per genotype). Significance tests compare proportion of cells in G1. ( A and E ) Data represent mean ± SEM. Significance determined by 1-way ANOVA with Tukey correction for multiple comparisons. * P <0.05, ** P <0.01, *** P < 0.001.

Article Snippet: Antibodies used for Western blots were: MYB (Abcam, ab45150), V5 (Cell Signaling Technology, 13202), ACTB (Santa Cruz Biotechnology Inc., sc-47778), GAPDH (Cell Signaling Technology, 2118), p16INK4A (Cell Signaling Technology, 29271), and p19ARF (Cell Signaling Technology, 77184).

Techniques: Derivative Assay, Transduction, Construct, Western Blot, Expressing, Cell Cycle Assay